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BRAF is the most frequently mutated gene in melanoma.,Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation.,Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy.,Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence.,In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells.,Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance.,Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation.,Transcriptomic data confirmed this hypothesis.,Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.

Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases.,Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra‐terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma.,Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF‐mutant melanoma.,We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF‐mutant melanoma cell lines.,Further, to investigate the molecular mechanisms underlying the effects of combined treatment, we conducted antibody arrays of in vitro drug‐treated cell lines and RNA sequencing of drug‐treated xenograft tumors.,The combination of JQ1 and Vemurafenib acted synergistically in BRAF‐mutant cell lines, resulting in marked apoptosis in vitro, with upregulation of proapoptotic proteins.,In vivo, combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone.,RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination, with several anti‐apoptotic genes significantly down‐regulated.,Collectively, our data provide a rationale for combined BET and BRAF inhibition as a novel strategy for the treatment of melanoma.

Mucosal melanoma is an aggressive melanoma with poor prognosis.,We assessed efficacy of pembrolizumab in patients with advanced mucosal melanoma in KEYNOTE-001 (NCT01295827), −002 (NCT01704287), and −006 (NCT01866319).,Patients received pembrolizumab 2 mg/kg every 3 weeks (Q3W) or 10 mg/kg Q2W or Q3W.,Response was assessed by independent central review per RECIST v1.1.,1567 patients were treated and 84 (5%) had mucosal melanoma.,Fifty-one of 84 were ipilimumab-naive.,In patients with mucosal melanoma, the objective response rate (ORR) was 19% (95% CI 11-29%), with median duration of response (DOR) of 27.6 months (range 1.1 + to 27.6).,Median progression-free survival (PFS) was 2.8 months (95% CI 2.7-2.8), with median overall survival (OS) of 11.3 months (7.7-16.6).,ORR was 22% (95% CI 11-35%) and 15% (95% CI 5-32%) in ipilimumab-naive and ipilimumab-treated patients.,Pembrolizumab provides durable antitumour activity in patients with advanced mucosal melanoma regardless of prior ipilimumab.

This phase I b study evaluated the safety and anti-tumor activity of pembrolizumab in Japanese patients with advanced melanoma.,Pembrolizumab (2 mg/kg) was given every 3 weeks (Q3W) for up to 2 years or until confirmed progression or unacceptable toxicity.,The tumor response was assessed as per the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) by both investigator review and central review.,Forty-two patients with advanced melanoma received pembrolizumab.,A primary cutaneous histology was observed in 34 patients (81.0%), while a primary mucosal histology was observed in 8 patients (19.0%).,Thirty-four patients (81.0%) experienced treatment-related adverse events (AEs).,The most common treatment-related AEs were pruritus, maculopapular rash, malaise, and hypothyroidism.,Grade 3-5 treatment-related AEs occurred in 8 patients (19.0%).,The only grade 3-5 treatment-related AE reported in at least two patients was anemia.,There were two treatment-related deaths (unknown cause and cerebral hemorrhage).,Among the 37 evaluable patients, the confirmed overall response rates (ORRs) determined by central review were 24.1% (95% CI 10.3-43.5) for cutaneous melanoma and 25.0% (95% CI 3.2-65.1) for mucosal melanoma.,The responses were durable, and the median duration of response was not reached in either population.,The median overall survival (OS) was not reached, with a 12-month OS of 82.7% for cutaneous melanoma and 51.4% for mucosal melanoma.,The safety profile of pembrolizumab in Japanese patients was similar to that reported in the previous clinical studies.,Pembrolizumab provided promising anti-tumor activity in Japanese patients with advanced melanoma.

Resistance to TRAIL (TNF-related apoptosis-inducing ligand)- induced apoptosis limits its therapeutic use.,Different strategies of TRAIL sensitization and a dependency on Bax have been reported, but common principles of TRAIL resistance and the way of Bax activation remained poorly understood.,Applying a melanoma model of TRAIL-sensitive and -resistant cell lines, efficient sensitization for TRAIL-induced apoptosis is demonstrated by the kinase inhibitor BMS-345541 (N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-1,2-ethanediamine hydrochloride), which targets IκB (inhibitor of κB proteins) kinase β (IKKβ).,This effect was completely abrogated by Bax knockout as well as by Bcl-2 overexpression, in accordance with a Bax dependency.,Early loss of the mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspases) clearly indicated the activation of mitochondrial apoptosis pathways.,Of note, BMS-345541 alone resulted in an early Bax activation, seen by conformational changes and by Bax translocation.,The synergistic effects can be explained by Bid activation through TRAIL, which inhibits Bcl-2, and the activation of Bax through BMS-345541.,The critical roles of XIAP (X-chromosome-linked inhibitor of apoptosis protein), Smac and Bid were clearly proven by overexpression and siRNA knockdown, respectively.,The way of Bax activation by BMS-345541 was unraveled by establishing new assays for Bax activation.,These showed reduction of the inactivating Bax phosphorylation at serine-184, while the activating Bax phosphorylation at threonine-167 was enhanced.,Thus, modulation of Bax phosphorylation appeared as tightly related to TRAIL sensitivity/resistance in melanoma cells, and therapeutic strategies may be considered.

Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers.,However, the optimal patient population who benefit from this treatment is unknown.,Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20.,Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment.,Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS.,Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS−), giving CBR rates of 52.9 vs 10%, P=0.041.,Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive.,The survival of ASS− patients receiving at least four doses at 320 IU m−2 was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024.,ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression.,Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.

The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts.,Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively.,The strong antimetastatic activity of MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10 melanoma lung metastases in drug-treated mice.,Our data also show that MC3181 affects the lactate production in the high glycolytic WM266.4 cell line.,To unveil the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the degree of activation of different MAPK pathways, as well as the expression/activity levels of several proteins involved in angiogenesis, invasion, and survival (i.e.,AP2, MCAM/MUC18, N-cadherin, VEGF and MMP-2).,Our data disclosed both a decrease of the phospho-active form of JNK and an increased expression of the transcription factor AP2, events that occur in the very early phase of drug treatment and may be responsible of the antimetastatic effects of MC3181.

To characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures.,Primary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures.,These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed.,In monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells.,In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids.,This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines.,Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures.,This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo.,This study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor.,Differences in the cell culture method does plays an important role in phospholipid composition of the cells.,The online version of this article (doi:10.1186/s40659-017-0117-8) contains supplementary material, which is available to authorized users.

Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases.,Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies.,In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance.,Thus, combined treatment targeting both pathways is a promising strategy to overcome this.,Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines.,Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis.,Finally, an in vivo treatment experiment was carried out on NOD/SCID mice.,We show that combined therapy was more effective than monotherapy.,Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo.,This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.

Alterations in transcriptional programs promote tumor development and progression and are targetable by bromodomain and extraterminal (BET) protein inhibitors.,However, in a multi‐site clinical trial testing the novel BET inhibitor, PLX51107, in solid cancer patients, liver metastases of uveal melanoma (UM) patients progressed rapidly following treatment.,Mechanisms of resistance to BET inhibitors in UM are unknown.,We show that fibroblast growth factor 2 (FGF2) rescued UM cells from growth inhibition by BET inhibitors, and FGF2 effects were reversible by FGF receptor (FGFR) inhibitors.,BET inhibitors also increased FGFR protein expression in UM cell lines and in patient tumor samples.,Hepatic stellate cells (HSCs) secrete FGF2, and HSC‐conditioned medium provided resistance of UM cells to BET inhibitors.,PLX51107 was ineffective in vivo, but the combination of a FGFR inhibitor, AZD4547, and PLX51107 significantly suppressed the growth of xenograft UM tumors formed from subcutaneous inoculation of UM cells with HSCs and orthotopically in the liver.,These results suggest that co‐targeting of FGFR signaling is required to increase the responses of metastatic UM to BET inhibitors.